Multiplex pcr detection of alk, ret, and ros fusions

ABSTRACT

Provided herein are methods and compositions for multiplex detection of a large number of actionable gene fusions with very high sensitivity and specificity. The present methods and compositions can detect ALK, RET, and ROS1 gene fusions, optionally in combination with other mutations and fusions.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 15/993,375, filed on May 30, 2018, which claims thebenefit of priority to US Provisional Application No. 62/513,226, filedon May 31, 2017, the content of each disclosure is incorporated byreference herein in its entirety.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronicST26 XML file named “34290_US2_Sequence_Listing.xml”, having a size inbytes of 255 kb, and created on Oct. 5, 2022, and is incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

A number of cancers are associated with gene fusions (Yoshihara et al.(2015) Oncogene 34:4845). Perhaps the earliest reported example is theassociation of BCR-ABL with chronic myelogenous leukemia (CML) in the′60s (Nowell and Hungerford (1960) J. Natl. Cancer Inst. 25:85). Sincethen, hundreds more gene fusions have been reported for cancers in manydifferent tissues (Presner and Chinnaiyan (2009) Curr. Opin Genet. Dev.19:82).

Another example is the tyrosine receptor kinase ALK (anaplastic lymphomakinase). EML4-ALK (echinoderm microtubule-associated protein-like4-anaplastic lymphoma kinase) fusions are associated with non-small celllung cancer (NSCLC). In this case, the N terminal, extracellular portionof ALK is replaced by EML4 (KIF5B, HIP1, KLC1, TFG can also fuse withALK in a similar manner). The expression of the resulting fusion gene isdriven by the strong EML4 promoter, resulting in higher expression ofthe intracellular tyrosine kinase domain of ALK. In addition, EML4 formsa coiled-coil that results in ligand-independent dimerization, andconstitutive activation of the ALK tyrosine kinase domain. Additionalexamples of activated kinase fusions involve RET (rearranged duringtransfection) and ROS1.

Detection of a gene fusion can be used to direct therapy. Most methodsof detection require biopsy of tumor tissue, which is not feasible formany cancer patients, especially in later stages. Detection in biopsiedtissue sections is typically carried out by fluorescence in situhybridization (FISH) or immunohistochemistry (IHC). The tests have highfalse positive rates and background, in part because of shearing duringthe sectioning process. Skilled cytologists are thus required to observemultiple tissue sections, which necessitates a sizable biopsy from aweakened patient. Similarly, a difficulty with using RT-PCR is theamount and quality of genetic material from tumor tissue, e.g., informalin fixed paraffin embedded (FFPE) form. See, e.g., Liu et al.(2015) PLoSOne 10: e0117032.

Because detection is time and resource intensive, the testing rate isrelatively low. Cancers associated with ALK fusions are very sensitiveto ALK inhibitors such as crizotinib and ceretinib. Gene fusions withRearranged during Transcription (RET), such as with KIF5B or CCDC6, arealso sensitive to therapy, e.g., with vandetanib (see Matsubara et al.(2007) J. Thorac. Oncol. 7:1872). The low rate of testing for genefusions thus represents a great lost opportunity for treatment.

SUMMARY OF THE INVENTION

Provided herein are multiplex methods and compositions for detectingfusion genes, in particular those involving ALK, RET, and ROS1.

Provided herein are multiplex assay compositions comprising: (A) atleast one primer set and labeled probe that specifically amplify anddetect at least one ALK fusion gene; (B) at least one primer set andlabeled probe that specifically amplify and detect at least one RETfusion gene; (C) at least one primer set and labeled probe thatspecifically amplify and detect at least one ROS1 fusion gene; and (D) aprimer set and labeled probe that specifically amplify and detect aninternal control. Further provided are multiplex assay compositionscomprising: (A) at least one primer set and labeled probe thatspecifically amplify and detect at least one ALK fusion gene; (B) atleast one primer set and labeled probe that specifically amplify anddetect at least one RET fusion gene; and (C) a primer set and labeledprobe that specifically amplify and detect an internal control. Providedherein are multiplex assay compositions comprising: (A) at least oneprimer set and labeled probe that specifically amplify and detect atleast one RET fusion gene; and (B) a primer set and labeled probe thatspecifically amplify and detect an internal control.

In some embodiments, the at least one ALK fusion gene is selected fromthe group consisting of: EML4 exon 13-ALK exon 20, EML4 exon 20-ALK exon20, EML4 exon 6a/b-ALK exon 20, EML4 exon 2-ALK exon 20, EML4 exon18-ALK exon 20, KIF5B exon 17-ALK exon 20, and KIF5B exon 24-ALK exon20; the at least one RET fusion gene is selected from the groupconsisting of: KIF5B exon 15-RET exon 12, KIF5B exon 16-RET exon 12,KIF5B exon 22-RET exon 12, KIF5B exon 23-RET exon 12, CCDC6 exon 1-RETexon 12, and NCOA4 exon 6-RET exon 12; and the at least one ROS1 fusiongene is selected from the group consisting of: CD74 exon 6-ROS1 exon 34,CD74 exon 6-ROS1 exon 32, EZR exon 10-ROS1 exon 34, TPM3 exon 8-ROS1exon 35, SDC4 exon 4-ROS1 exon 32, SDC4 exon 2-ROS1 exon 32, SDC4 exon2-ROS1 exon 34, SDC4 exon 4-ROS1 exon 34, SLC34A2 exon 13-ROS1 exon 34,SLC34A2 exon 13-ROS1 exon 32, SLC34A2 exon 4-ROS1 exon 32, SLC34A2 exon4-ROS1 exon 35, and LRIG3 exon 16-ROS1 exon 35, in any combination.

In some embodiments, the composition comprises at least one primer setand probe that amplify and detect more than 2 ALK fusion genes, morethan 2 RET fusion genes, and/or more than 2 ROS1 fusion genes. In someembodiments, the composition comprises at least one primer set and probethat amplify and detect EML4 exon 13-ALK exon 20, EML4 exon 20-ALK exon20, EML4 exon 6a/b-ALK exon 20, KIF5B exon 15-RET exon 12, KIF5B exon16-RET exon 12, KIF5B exon 22-RET exon 12, CD74 exon 6-ROS1 exon 34, andEZR exon 10-ROS1 exon 34.

In some embodiments, the at least one ALK fusion gene include: EML4 exon13-ALK exon 20, EML4 exon 20-ALK exon 20, EML4 exon 6a/b-ALK exon 20,EML4 exon 2-ALK exon 20, EML4 exon 18-ALK exon 20, KIF5B exon 17-ALKexon 20, and KIF5B exon 24-ALK exon 20; the at least one RET fusion geneincludes: KIF5B exon 15-RET exon 12, KIF5B exon 16-RET exon 12, KIF5Bexon 22-RET exon 12, KIF5B exon 23-RET exon 12, CCDC6 exon 1-RET exon12, and NCOA4 exon 6-RET exon 12; and the at least one ROS1 fusion geneincludes: CD74 exon 6-ROS1 exon 34, CD74 exon 6-ROS1 exon 32, EZR exon10-ROS1 exon 34, TPM3 exon 8-ROS1 exon 35, SDC4 exon 4-ROS1 exon 32,SDC4 exon 2-ROS1 exon 34, SDC4 exon 2-ROS1 exon 32, SDC4 exon 4-ROS1exon 32, SLC34A2 exon 13-ROS1 exon 34, SLC34A2 exon 13-ROS1 exon 32,SLC34A2 exon 4-ROS1 exon 32, SLC34A2 exon 4-ROS1 exon 35, and LRIG3 exon16-ROS1 exon 35. That is, the assay composition includes primer sets andprobes to amplify and detect all of the listed fusion genes.

In some embodiments, for the primer set to amplify at least one ALKfusion gene, the forward primer and reverse primer have sequencesselected from the group consisting of SEQ ID NOs:1-50, and SEQ IDNOs:52-61 and 181, respectively. In some embodiments, for the probe todetect at least one ALK fusion gene, the probe sequence is selected fromthe group consisting of SEQ ID NOs:182-186. The forward and reverseprimer sequences and probe sequences can be used together in anyappropriate combination to detect any 1, 2, 3, 4, 5, 6, or 7 ALK fusionvariants in any combination. In some embodiments, for the primer set toamplify at least one RET fusion gene, the forward primer and reverseprimer have sequences selected from the group consisting of SEQ IDNOs:83-145 and 187, and SEQ ID NOs:161-180, respectively. In someembodiments, for the probe to detect at least one RET fusion gene, theprobe sequence is selected from the group consisting of:189-194. Theforward and reverse primer sequences and probe sequences can be usedtogether in any combination to detect any 1, 2, 3, 4, 5, or 6 RET fusionvariants in any combination. In some embodiments, for the primer set todetect at least one ROS1 fusion gene, the forward primer and reverseprimer have sequences selected from the group consisting of SEQ IDNOs:195-212, and SEQ ID NOs:213-226, respectively. In some embodiments,for the probe to detect at least one ROS1 fusion gene, the probesequence is selected from the group consisting of:227-230 and 51. Theforward and reverse primer sequences and probe sequences can be usedtogether in any combination to detect any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, or 13 ROS1 fusion variants in any combination.

In some embodiments, the label on labeled probe that detects theinternal control is different from the labels on the labeled probes thatdetect the fusion genes. In some embodiments, the labels on all of thelabeled probes are different from each other. In some embodiments, asingle labeled probe is used to detect all of the at least one ALKfusion genes. In some embodiments, a single labeled probe is used todetect all of the at least one RET fusion genes. In some embodiments, asingle labeled probe is used to detect all of the at least one ROS1fusion genes. In some embodiments, the labeled probe is attached to aprimer in the at least one primer set. In some embodiments, the labeledprobe is separate from the primer set.

In some embodiments, where more than one ALK fusion gene is amplifiedand detected, all of the primer sets that amplify the ALK fusion genesinclude a single common primer. In some embodiments, where more than oneALK fusion gene is amplified and detected, the primer sets includeunique primers. In some embodiments, where more than one RET fusion geneis amplified and detected, all of the primer sets that amplify the RETfusion genes include a single common primer. In some embodiments, wheremore than one RET fusion gene is amplified and detected, the primer setsinclude unique primers. In some embodiments, where more than one ROS1fusion gene is amplified and detected, all of the primer sets thatamplify the ROS1 fusion genes include a single common primer. In someembodiments, where more than one ROS1 fusion gene is amplified anddetected, the primer sets include unique primers.

Further provided herein are multiplex assay compositions comprising: (A)at least one primer set and labeled probe that specifically amplify anddetect at least one ALK fusion gene; (B) at least one primer set andlabeled probe that specifically amplify and detect at least one RETfusion gene; and (C) a primer set and labeled probe that specificallyamplify and detect an internal control. Also provided herein aremultiplex assay compositions comprising: (A) at least one primer set andlabeled probe that specifically amplify and detect at least one RETfusion gene; and (B) a primer set and labeled probe that specificallyamplify and detect an internal control. In some embodiments, at leastone ROS1 fusion gene is amplified and detected in a separate multiplexassay. In some embodiments, the at least one ALK fusion gene is selectedfrom the group consisting of: EML4 exon 13-ALK exon 20, EML4 exon 20-ALKexon 20, EML4 exon 6a/b-ALK exon 20, EML4 exon 2-ALK exon 20, EML4 exon18-ALK exon 20, KIF5B exon 17-ALK exon 20, and KIF5B exon 24-ALK exon20; and the at least one RET fusion gene is selected from the groupconsisting of: KIF5B exon 15-RET exon 12, KIF5B exon 16-RET exon 12,KIF5B exon 22-RET exon 12, KIF5B exon 23-RET exon 12, CCDC6 exon 1-RETexon 12, and NCOA4 exon 6-RET exon 12, in any combination. In someembodiments, the at least one ROS1 fusion gene is selected from thegroup consisting of: CD74 exon 6-ROS1 exon 34, CD74 exon 6-ROS1 exon 32,EZR exon 10-ROS1 exon 34, TPM3 exon 8-ROS1 exon 35, SDC4 exon 2-ROS1exon 34, SDC4 exon 4-ROS1 exon 32, SDC4 exon 2-ROS1 exon 32, SDC4 exon4-ROS1 exon 34, SLC34A2 exon 13-ROS1 exon 34, SLC34A2 exon 13-ROS1 exon32, SLC34A2 exon 4-ROS1 exon 32, SLC34A2 exon 4-ROS1 exon 35, and LRIG3exon 16-ROS1 exon 35.

In some embodiments, the composition comprises at least one primer setand probe that amplify and detect more than 2 ALK fusion genes and morethan 2 RET fusion genes. In some embodiments, the composition comprisesat least one primer set and probe that amplify and detect EML4 exon13-ALK exon 20, EML4 exon 20-ALK exon 20, EML4 exon 6a/b-ALK exon 20,KIF5B exon 15-RET exon 12, KIF5B exon 16-RET exon 12, and KIF5B exon22-RET exon 12.

In some embodiments, the at least one ALK fusion gene include: EML4 exon13-ALK exon 20, EML4 exon 20-ALK exon 20, EML4 exon 6a/b-ALK exon 20,EML4 exon 2-ALK exon 20, EML4 exon 18-ALK exon 20, KIF5B exon 17-ALKexon 20, and KIF5B exon 24-ALK exon 20; and the at least one RET fusiongene includes: KIF5B exon 15-RET exon 12, KIF5B exon 16-RET exon 12,KIF5B exon 22-RET exon 12, KIF5B exon 23-RET exon 12, CCDC6 exon 1-RETexon 12, and NCOA4 exon 6-RET exon 12.

In some embodiments, the label on labeled probe that detects theinternal control is different from the labels on the labeled probes thatdetect the fusion genes. In some embodiments, the labels on all of thelabeled probes are different from each other. In some embodiments, asingle labeled probe is used to detect all of the at least one ALKfusion genes. In some embodiments, a single labeled probe is used todetect all of the at least one RET fusion genes. In some embodiments,the labeled probe is attached to a primer in the at least one primerset. In some embodiments, the labeled probe is separate from the primerset.

In some embodiments, where more than one ALK fusion gene is amplifiedand detected, all of the primer sets that amplify the ALK fusion genesinclude a single common primer. In some embodiments, where more than oneALK fusion gene is amplified and detected, the primer sets includeunique primers. In some embodiments, where more than one RET fusion geneis amplified and detected, all of the primer sets that amplify the RETfusion genes include a single common primer. In some embodiments, wheremore than one RET fusion gene is amplified and detected, the primer setsinclude unique primers.

Examples of internal controls that can be used for the presentlydisclosed assays include, but are not limited to, SDHA (succinatedehydrogenase), LDHA (lactate dehydrogenase A), NONO, PGK(phosphoglycerate kinase 1), PPIH, HPRT1, beta-actin, GADPH, ACTB, and16S rRNA.

In some embodiments, the composition further comprises a DNA polymerase,e.g., a thermostable DNA polymerase such as Taq or a Taq derivative. Insome embodiments, the composition further comprises reversetranscriptase. In some embodiments, the composition further comprisesdNTPs. In some embodiments, the composition further comprises bufferamenable to polymerization by the DNA polymerase and reversetranscriptase.

In some embodiments, the composition further comprises a biologicalsample from an individual or group of individuals. In some embodiments,the individual has been diagnosed with cancer, e.g., lung cancer (e.g.,non-small cell lung cancer (NSCLC), lung squamous cell carcinoma, lungadenocarcinoma), bladder carcinoma, glioblastoma, head and neck cancer,glioma, thyroid carcinoma, ovarian cancer, leukemia, lymphoma, prostatecancer, pancreatic cancer, renal cancer, or breast cancer.

In some embodiments, the sample is enriched or isolated nucleic acid,e.g., DNA or RNA. In some embodiments, the sample is RNA, e.g., isolatedfrom blood (e.g., serum, plasma, other blood fraction), bronchoalveolarlavage, or tissue biopsy. In some embodiments, the biological sampleincludes 100 nM or less of the polynucleotide comprising the fusiongene, e.g., 0.01-100 nM, 0.01-25 nM, 0.01-5 nM, 0.02-0.5 nM, or 0.02-0.1nM.

Further provided are methods of identifying an individual with cancercomprising contacting a biological sample from the individual with anyof the multiplex assay compositions described herein (e.g., comprising:(A) at least one primer set and labeled probe that specifically amplifyand detect at least one ALK fusion gene; (B) at least one primer set andlabeled probe that specifically amplify and detect at least one RETfusion gene; (C) at least one primer set and labeled probe thatspecifically amplify and detect at least one ROS1 fusion gene; and (D) aprimer set and labeled probe that specifically amplify and detect aninternal control); carrying out amplification and detection underconditions that allow formation and detection of an amplificationproduct in the presence of at least one fusion gene in the biologicalsample; determining that at least one fusion gene is present if a fusiongene is detected in step B; whereby the presence of at least one fusiongene in said individual's sample indicates sensitivity of saidindividual to a kinase inhibitor therapy if at least one fusion gene ispresent.

Further provided are methods of determining the likelihood of responseof an individual with cancer to kinase therapy comprising contacting abiological sample from the individual with any of the multiplex assaycompositions described herein (e.g., comprising: (A) at least one primerset and labeled probe that specifically amplify and detect at least oneALK fusion gene; (B) at least one primer set and labeled probe thatspecifically amplify and detect at least one RET fusion gene; (C) atleast one primer set and labeled probe that specifically amplify anddetect at least one ROS1 fusion gene; and (D) a primer set and labeledprobe that specifically amplify and detect an internal control);carrying out amplification and detection under conditions that allowformation and detection of an amplification product in the presence ofat least one fusion gene in the biological sample; determining that atleast one fusion gene is present if a fusion gene is detected in step B;and determining that the individual will likely respond to the kinaseinhibitor therapy.

Further provided are methods of treating an individual, e.g., anindividual diagnosed with cancer, comprising contacting a biologicalsample from the individual with any of the multiplex assay compositionsdescribed herein (e.g., comprising: (A) at least one primer set andlabeled probe that specifically amplify and detect at least one ALKfusion gene; (B) at least one primer set and labeled probe thatspecifically amplify and detect at least one RET fusion gene; (C) atleast one primer set and labeled probe that specifically amplify anddetect at least one ROS1 fusion gene; and (D) a primer set and labeledprobe that specifically amplify and detect an internal control);carrying out amplification and detection under conditions that allowformation and detection of an amplification product in the presence ofat least one fusion gene in the biological sample; determining that atleast one fusion gene is present if a fusion gene is detected; andtreating the individual if at least one fusion gene is present. Furtherprovided are methods of treating an individual, e.g., an individualdiagnosed with cancer, comprising contacting a biological sample fromthe individual with any of the multiplex assay compositions describedherein (e.g., comprising: (A) at least one primer set and labeled probethat specifically amplify and detect at least one ALK fusion gene; (B)at least one primer set and labeled probe that specifically amplify anddetect at least one RET fusion gene; and (C) a primer set and labeledprobe that specifically amplify and detect an internal control);carrying out amplification and detection under conditions that allowformation and detection of an amplification product in the presence ofat least one fusion gene in the biological sample; determining that atleast one fusion gene is present if a fusion gene is detected; andtreating the individual if at least one fusion gene is present. Furtherprovided are methods of treating an individual, e.g., an individualdiagnosed with cancer, comprising contacting a biological sample fromthe individual with any of the multiplex assay compositions describedherein (e.g., comprising: (A) at least one primer set and labeled probethat specifically amplify and detect at least one RET fusion gene; and(B) a primer set and labeled probe that specifically amplify and detectan internal control); carrying out amplification and detection underconditions that allow formation and detection of an amplificationproduct in the presence of at least one fusion gene in the biologicalsample; determining that at least one fusion gene is present if a fusiongene is detected; and treating the individual if at least one fusiongene is present.

In some embodiments, the treatment is with a kinase inhibitor, e.g., aselective kinase inhibitor such as alectinib, crizotinib, ceritinib,lorlatinib, brigatinib, cabozantinib, apatinib, vandetanib, ponatinib,lenvatinib, DS6051b, or variants or combinations thereof. In someembodiments, the course of treatment includes radiation therapy orchemotherapy (e.g., cisplatin, carboplatin, paclitaxel, docetaxel). Insome embodiments, the treatment is with GSK1838705A, TAE-684, CEP-14083,AP26113, NMS-E628, sorafenib, vandetanib, motesanib, sunitinib, andXL-184 (see, e.g., Mologni (2011) Curr. Med. Chem. 18:162).

In some embodiments, the individual is monitored throughout treatment,e.g., to determine if the amount of fusion gene amplification productincreases or decreases, or if a different fusion gene is detected. Insome embodiments, the treatment is changed if the amount of fusion geneamplification product changes, or if a different fusion gene isdetected. For example, if the amount of the originally detected fusiongene decreases but the cancer is progressing, treatment can be changedto be less targeted, e.g., radio- or chemotherapy. If the individual'scondition has improved, treatment can be reduced.

In some embodiments, the biological sample includes DNA or RNA, e.g.,separated or purified nucleic acids. In some embodiments, the biologicalsample is RNA from blood, e.g., plasma, serum, or other blood fraction.In some embodiments, the amplification and detection are carried outusing qRT-PCR.

In some embodiments, the individual is diagnosed with lung cancer (e.g.,non-small cell lung cancer (NSCLC), lung squamous cell carcinoma, lungadenocarcinoma), bladder carcinoma, glioblastoma, head and neck cancer,glioma, thyroid carcinoma, ovarian cancer, leukemia, lymphoma, prostatecancer, pancreatic cancer, renal cancer, or breast cancer.

Further provided are methods for determining the presence of at leastone fusion gene in a sample from an individual, e.g., an individualdiagnosed with cancer, comprising contacting a biological sample fromthe individual with any of the multiplex assay compositions describedherein (e.g., comprising: (A) at least one primer set and labeled probethat specifically amplify and detect at least one ALK fusion gene; (B)at least one primer set and labeled probe that specifically amplify anddetect at least one RET fusion gene; (C) at least one primer set andlabeled probe that specifically amplify and detect at least one ROS1fusion gene; and (D) a primer set and labeled probe that specificallyamplify and detect an internal control); carrying out amplification anddetection under conditions that allow formation and detection of anamplification product in the presence of at least one fusion gene in thebiological sample; determining that at least one fusion gene is presentif a fusion gene is detected. Further provided are methods fordetermining the presence of at least one fusion gene in a sample from anindividual, e.g., an individual diagnosed with cancer, comprisingcontacting a biological sample from the individual with any of themultiplex assay compositions described herein (e.g., comprising: (A) atleast one primer set and labeled probe that specifically amplify anddetect at least one ALK fusion gene; (B) at least one primer set andlabeled probe that specifically amplify and detect at least one RETfusion gene; and (C) a primer set and labeled probe that specificallyamplify and detect an internal control); carrying out amplification anddetection under conditions that allow formation and detection of anamplification product in the presence of at least one fusion gene in thebiological sample; determining that at least one fusion gene is presentif a fusion gene is detected. Further provided are methods fordetermining the presence of at least one fusion gene in a sample from anindividual, e.g., an individual diagnosed with cancer, comprisingcontacting a biological sample from the individual with any of themultiplex assay compositions described herein (e.g., comprising: (A) atleast one primer set and labeled probe that specifically amplify anddetect at least one RET fusion gene; and (B) a primer set and labeledprobe that specifically amplify and detect an internal control);carrying out amplification and detection under conditions that allowformation and detection of an amplification product in the presence ofat least one fusion gene in the biological sample; determining that atleast one fusion gene is present if a fusion gene is detected.

In some embodiments, the biological sample includes DNA or RNA, e.g.,separated or purified nucleic acids. In some embodiments, the biologicalsample is RNA from blood, e.g., plasma, serum, or other blood fraction.In some embodiments, the amplification and detection are carried outusing qRT-PCR.

In some embodiments, the individual is diagnosed with lung cancer (e.g.,non-small cell lung cancer (NSCLC), lung squamous cell carcinoma, lungadenocarcinoma), bladder carcinoma, glioblastoma, head and neck cancer,glioma, thyroid carcinoma, ovarian cancer, leukemia, lymphoma, prostatecancer, pancreatic cancer, renal cancer, or breast cancer.

In some embodiments, the method further comprises determining a courseof treatment if at least one fusion gene is detected. In someembodiments, the treatment is with a kinase inhibitor, e.g., a selectivekinase inhibitor such as alectinib, crizotinib, ceritinib, lorlatinib,brigatinib, cabozantinib, apatinib, vandetanib, ponatinib, lenvatinib,DS6051b, or variants or combinations thereof. In some embodiments, thecourse of treatment includes radiation therapy or chemotherapy (e.g.,cisplatin, carboplatin, paclitaxel, docetaxel). In some embodiments, thetreatment is with GSK1838705A, TAE-684, CEP-14083, AP26113, NMS-E628,sorafenib, vandetanib, motesanib, sunitinib, and XL-184.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows that the indicated ALK fusion variants (FAM) aredetectable at 50 copies in 0.1 ng WT RNA (n=3). FIG. 1B shows that theindicated RET fusion variants (HEX) are detectable at 50 copies in 0.1ng WT RNA (n=3). FIG. 1C shows that the indicated ROS1 fusion variants(JA270) are detectable at 50 copies in 0.1 ng WT RNA (n=3). FIG. 1Dshows the internal control Ct values for each input RNA.

FIG. 2 shows the limit of detection of ALK and RET fusions in amultiplex assay as described herein. The assay is able to detect 25copies of fusion transcript diluted in UHR.

FIG. 3 shows linearity data for representative ALK fusion variants

FIG. 4 shows linearity data for representative RET fusion variants.

FIG. 5 shows linearity data for representative ROS1 fusion variants

FIG. 6 shows LOD data for a representative ALK fusion variant.

FIG. 7 shows LOD data for a representative RET fusion variant.

FIG. 8 shows LOD data for a representative ROS1 fusion variant.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The inventors have discovered a novel, quantitative, and multiplexmethod of detecting fusions between genetic regions. The presentlydisclosed methods require only a small amount of patient sample that canbe gathered non-invasively, e.g., circulating free RNA (cfRNA) fromplasma.

Current tests require either biopsy or large amounts of plasma, due tothe limited amount of circulating nucleic acids originating from atumor. The presently described methods allow for an extremely sensitive(down to ˜25 copies), one-tube assay to detect multiple gene fusionsthat are predictive of cancer and response to therapy. The presentassays can be used for identification of a fusion variant, as well asmonitoring and surveillance during treatment and/or progression.

II. Definitions

A “genetic fusion” is hybrid chromosomal sequence formed by joining oftwo chromosomal locations that were previously separate. Fusion canoccur between genes on the same chromosome (e.g., interstitial deletionor chromosomal inversion) or on different chromosomes (e.g.,translocation).

A “fusion gene” is a hybrid gene formed by the joining of two genes thatwere previously separate, leading to a structural rearrangement and/orvariant in the tumor genome. The fusion gene need not necessarilyinclude coding sequence from both genes, but can include non-codingsequence from one of the genes, e.g., promoter or 3′ untranslatedregions. The denomination of genes that comprise a fusion gene as “gene1,” “gene 2,” “gene A,” “gene B,” etc., is used to distinguish betweengenes that make up the fusion and does not necessarily refer to theposition of the genes in the fusion. The terms ALK fusion, RET fusion,and ROS1 fusion refer to fusion genes that include ALK, RET, and ROS1 asa member, respectively.

The terms “fusion site,” “fusion point,” “breakpoint” and like termsrefer to the point in a genetic fusion where a nucleotide from one geneor genetic location is found adjacent to a nucleotide from another geneor genetic location.

The terms “target region,” “target portion,” “target fragment,” and liketerms refer to a region of a target nucleic acid sequence that is to beamplified and/or analyzed.

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” referto polymers of nucleotides (e.g., ribonucleotides ordeoxyribo-nucleotides) and includes naturally-occurring (adenosine,guanidine, cytosine, uracil and thymidine), non-naturally occurring, andmodified nucleic acids. The term is not limited by length (e.g., numberof monomers) of the polymer. A nucleic acid may be single-stranded ordouble-stranded and will generally contain 5′-3′ phosphodiester bonds,although in some cases, nucleotide analogs may have other linkages.Monomers are typically referred to as nucleotides. The term “non-naturalnucleotide” or “modified nucleotide” refers to a nucleotide thatcontains a modified nitrogenous base, sugar or phosphate group, or thatincorporates a non-natural moiety in its structure. Examples ofnon-natural nucleotides include dideoxynucleotides, biotinylated,aminated, deaminated, alkylated, benzylated and fluorophor-labelednucleotides.

The term “primer” refers to a short nucleic acid (an oligonucleotide)that acts as a point of initiation of polynucleotide strand synthesis bya nucleic acid polymerase under suitable conditions. Polynucleotidesynthesis and amplification reactions typically include an appropriatebuffer, dNTPs and/or rNTPs, and one or more optional cofactors, and arecarried out at a suitable temperature. A primer typically includes atleast one target-hybridized region that is at least substantiallycomplementary to the target sequence. This region of is typically about15 to about 40 nucleotides in length. A “primer pair” refers to aforward primer and reverse primer (sometimes called 5′ and 3′ primers)that are complementary to opposite strands of a target sequence anddesigned to amplify the target sequence. The forward and reverse primersare arranged within an amplifiable distance of each other on the targetsequence, e.g., about 10-5000 nucleotides, about 25-500, or about 60-120nucleotides. A “primer set” refers to one or more primer pairs, or acombination of at least one forward primer and at least one reverseprimer. For example, a primer set can include 3 forward primers and 1reverse primer, so that 3 distinct amplification products canpotentially be produced.

A primer set or primer pair that is specific for a sequence (or portionof a gene) that is 5′ (or 3′) of a fusion site (or breakpoint) refers toprimers used to amplify a sequence that does not include the fusion siteor breakpoint.

As used herein, “probe” means any molecule that is capable ofselectively binding to a specifically intended target biomolecule, forexample, a nucleic acid sequence of interest to be bound, captured orhybridized by the probes. Probes are typically labeled with anon-naturally occurring moiety, e.g., a fluorophore, chromophore,affinity tag (e.g., streptavidin or biotin), and/or a quencher.

The words “complementary” or “complementarity” refer to the ability of anucleic acid in a polynucleotide to form a base pair with anothernucleic acid in a second polynucleotide. For example, the sequence A-G-T(A-G-U for RNA) is complementary to the sequence T-C-A (U-C-A for RNA).Complementarity may be partial, in which only some of the nucleic acidsmatch according to base pairing, or complete, where all the nucleicacids match according to base pairing. A probe or primer is considered“specific for” a target sequence if it is at least partiallycomplementary to the target sequence. Depending on the conditions, thedegree of complementarity to the target sequence is typically higher fora shorter nucleic acid such as a primer (e.g., greater than 80%, 90%,95%, or higher) than for a longer sequence.

The terms “identical” or “percent identity,” in the context of two ormore nucleic acids, or two or more polypeptides, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides, or amino acids, that are the same (e.g.,about 60% identity, e.g., at least any of 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region, when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parameters, orby manual alignment and visual inspection. See e.g., the NCBI web siteat ncbi.nlm.nih.gov/BLAST. Such sequences are then said to be“substantially identical.” Percent identity is typically determined overoptimally aligned sequences, so that the definition applies to sequencesthat have deletions and/or additions, as well as those that havesubstitutions. The algorithms commonly used in the art account for gapsand the like. Typically, identity exists over a region comprising an asequence that is at least about 8-25 amino acids or nucleotides inlength, or over a region that is 50-100 amino acids or nucleotides inlength, or over the entire length of the reference sequence.

The term “allele” refers to a sequence variant of a gene. One or moregenetic differences can constitute an allele.

The term “kit” refers to any manufacture (e.g., a package or acontainer) including at least one reagent, such as a nucleic acid probeor probe pool or the like, for specifically amplifying, capturing,tagging/converting or detecting RNA or DNA as described herein.

The term “amplification conditions” refers to conditions in a nucleicacid amplification reaction (e.g., PCR amplification) that allow forhybridization and template-dependent extension of the primers. The terms“amplicon” and “amplification product” refer to a nucleic acid moleculethat contains all or a fragment of the target nucleic acid sequence andthat is formed as the product of in vitro amplification by any suitableamplification method. The borders of a given amplicon are typicallydefined by the position of the complementary portion of the forward andreverse primers used for amplification. Suitable PCR conditions aredescribed in PCR Strategies (Innis et al., 1995, Academic Press, SanDiego, Calif.) at Chapter 14; PCR Protocols: A Guide to Methods andApplications (Innis et al., Academic Press, N Y, 1990) The term“thermostable nucleic acid polymerase” or “thermostable polymerase”refers to a polymerase enzyme, which is relatively stable at elevatedtemperatures when compared, for example, to polymerases from E. coli. Athermostable polymerase is suitable for use under temperature cyclingconditions typical of the polymerase chain reaction (“PCR”). Exemplarythermostable polymerases include those from Thermus thermophilus,Thermus caldophilus, Thermus sp. Z05 (see, e.g., U.S. Pat. No.5,674,738) and mutants of the Thermus sp. Z05 polymerase, Thermusaquaticus, Thermus flavus, Thermus flliformis, Thermus sp. sps17,Deinococcus radiodurans, Hot Spring family B/clone 7, Bacillusstearothermophilus, Bacillus caldotenax, Thermotoga maritima, Thermotoganeapolitana and Thermosipho africanus, and modified versions thereof.

The term “sample” or “biological sample” refers to any compositioncontaining or presumed to contain nucleic acid from an individual. Theterm includes purified or separated components of cells, tissues, orblood, e.g., DNA, RNA, proteins, cell-free portions, or cell lysates. Insome embodiments, analysis is conducted on plasma samples isolated fromblood; the terms “detected in patient's blood” and “detected inpatient's plasma” are used interchangeably to mean that blood isobtained from the patient and plasma derived therefrom is used for theanalysis. A sample can also refer to other types of biological samples,e.g., skin, plasma, serum, whole blood and blood components (e.g.,platelets, buffy coat), saliva, urine, tears, seminal fluid, vaginalfluids, tissue biopsies, and other fluids and tissues, includingparaffin embedded tissues. Samples also may include constituents andcomponents of in vitro cultures of cells obtained from an individual,including cell lines.

A “control” sample or value refers to a sample that serves as areference, usually a known reference, for comparison to a test sample ortest conditions. For example, a test sample can be taken from a testcondition, e.g., from an individual suspected of having cancer, andcompared to samples from known conditions, e.g., from a cancer-freeindividual (negative control), or from an individual known to havecancer and/or a particular genetic abnormality (positive control). Inthe context of the present disclosure, an example of a negative controlwould be a biological sample from a known healthy (non-cancer,non-mutated) individual, and an example of a positive control would be abiological sample from a patient or cell line known to have a particulargene fusion. A control can also represent an average value or a rangegathered from a number of tests or results. A control can also beprepared for reaction conditions. For example, a positive control forthe presence of nucleic acid could include primers or probes that willdetect a sequence known to be present in the sample, while a negativecontrol would be free of nucleic acids. One of skill in the art willrecognize that controls can be designed for assessment of any number ofparameters. For example, a control can be devised to compare therapeuticbenefit based on pharmacological data (e.g., half-life) or therapeuticmeasures (e.g., comparison of benefit and/or side effects). Controls canbe designed for in vitro applications. One of skill in the art willunderstand which controls are valuable in a given situation and be ableto analyze data based on comparisons to control values. Controls arealso valuable for determining the significance of data. For example, ifvalues for a given parameter are widely variant in controls, variationin test samples will not be considered as significant.

An “internal control” (IC) refers to a nucleic acid that is expected tobe present in the sample, such as a housekeeping gene that is expressedor present at a fairly standard level across samples. The internalcontrol can be used to standardize the amount and quality of nucleicacid in the sample with that of other samples and ensure that theamplification and detection reaction is functioning. Examples ofinternal controls include SDH (succinate dehydrogenase), LDHA (lactatedehydrogenase A), NONO, PGK (phosphoglycerate kinase 1), PPIH, HPRT1,beta-actin, GADPH, ACTB, and 16S rRNA.

The term “diagnosis” refers to a relative probability that a subject hasa disorder such as cancer or certain type of cancer (e.g., resultingfrom a gene fusion). Similarly, the term “prognosis” refers to arelative probability that a certain future outcome may occur in thesubject. For example, in the context of the present disclosure,diagnosis can refer to classification of a cancer or the likelihood thatan individual will be responsive to a particular therapy. The terms arenot intended to be absolute, as will be appreciated by any one of skillin the field of medical diagnostics.

The terms “response to therapy,” “response to treatment,”“amelioration,” and like terms refer to any reduction in the severity ofsymptoms. In the case of treating cancer, treatment can refer to, e.g.,reducing tumor size, number of cancer cells, growth rate, metastaticactivity, reducing cell death of non-cancer cells, reduced nausea andother chemotherapy or radiotherapy side effects, etc. The terms “treat”and “prevent” are not intended to be absolute terms. Treatment andprevention can refer to any delay in onset, amelioration of symptoms,improvement in patient survival, increase in survival time or rate, etc.Treatment and prevention can be complete (undetectable levels ofneoplastic cells) or partial, such that fewer neoplastic cells are foundin a patient than would have occurred without the treatment. The effectof treatment can be compared to an individual or pool of individuals notreceiving the treatment (e.g., individuals having the same geneticfusion), or to the same patient prior to treatment or at a differenttime during treatment. In some aspects, the severity of disease isreduced by at least 10%, as compared, e.g., to the individual beforeadministration or to a control individual not undergoing treatment. Insome aspects the severity of disease is reduced by at least 25%, 50%,75%, 80%, or 90%, or in some cases, no longer detectable using standarddiagnostic techniques.

The terms “treat” and “administer,” with reference to a patient, includerecommending, providing, or prescribing a particular treatment to thepatient, and are not limited to directly, physically treating thepatient.

The term “threshold cycle” or “Ct” is a measure of relativeconcentration and is commonly used in real-time PCR (also referred to asqPCR). Ct refers to the intersection of an amplification curve and athreshold line. The threshold line is often set at a point when signalcan be detected above background, or when an amplification reactionenters the exponential phase. Ct can be affected by concentration oftarget and amplification conditions, e.g., the effect of conditions ondetectable labels and amplification efficiency. A higher Ct correspondsto a longer time to reach the threshold, be it due to low targetconcentration or inefficient amplification.

The terms “individual,” “subject,” “patient,” and like terms are usedinterchangeably and refer to humans, except where indicated. Othermammals can be considered subjects, such as non-human primates, as wellas rabbits, rats, mice, dogs, cats, and other mammalian species. Theterm does not necessarily indicate that the subject has been diagnosedwith a particular disease, but typically refers to an individual undermedical supervision. A patient can be seeking treatment, monitoring,adjustment or modification of an existing therapeutic regimen, etc. Apatient can include individuals that have not received treatment, arecurrently receiving treatment, have had surgery, and those that havediscontinued treatment.

The terms “label,” “tag,” “detectable moiety,” and like terms refer to acomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, chemical, or other physical means. For example, usefullabels include fluorescent dyes, luminescent agents, radioisotopes(e.g., ³²P, ³H), electron-dense reagents, or an affinity-based moiety,e.g., a “His tag” for purification, or a “strepavidin tag” thatinteracts with biotin.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Pfaffl, Methods: The ongoing evolution ofqPCR, vol. 50 (2010); van Pelt-Verkuil et al. Principles and TechnicalAspects of PCR Amplification, Springer (2010); Lackie, DICTIONARY OFCELL AND MOLECULAR BIOLOGY, Elsevier (4th ed. 2007); Sambrook et al.,MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (ColdSprings Harbor, N.Y. 1989). The term “a” or “an” is intended to mean“one or more.” The terms “comprise,” “comprises,” and “comprising,” whenpreceding the recitation of a step or an element, are intended to meanthat the addition of further steps or elements is optional and notexcluded.

III. Fusion Genes

A number of cancer-associated fusion genes are known, and appear in allmanner of cancers. Examples include lung cancer (e.g., non-small celllung cancer (NSCLC), lung squamous cell carcinoma, lung adenocarcinoma),bladder carcinoma, glioblastoma, head and neck cancer, glioma, thyroidcarcinoma, ovarian cancer, leukemia, lymphoma, prostate cancer,pancreatic cancer, renal cancer, and breast cancer. Cancer-associatedfusion genes commonly occur where one member of the fusion is a kinaseinvolved in a pro-growth signaling pathway, and the other membercontributes to elevated or constitutive expression or signaling. This isthe case for fusions of ALK, RET, and ROS1. Common fusion partners forALK are EML4 and KIF5B. Common fusion partners for RET are KIF5B, CCDC6,and NCOA4. Several genes are known to fuse with ROS1, including CD74,EZR, TPM3, SDC4, SLC34A2, and LRIG3 (see, e.g., Yoshihara et al. (2015)Oncogene 34:4845).

The present compositions and methods focus on design of multiplex assaysto detect ALK, RET, and ROS1 fusions. Invasive biopsy or excessive bloodcollection is often not feasible for cancer patients. The presentcompositions and methods allow for detection of several actionable genefusions with a relatively small sample from the patient, which can be anon-invasive plasma sample.

The design of these highly multiplexed assays can vary. Where multipleALK fusions are detected, for example, a common primer and probe thathybridize to sequences in the ALK gene near the fusion point, andprimers specific for various fusion partners, can be used. Thus, forexample, if 5 different ALK fusions are detected, the assay can include15 oligonucleotides (10 primers and 5 probes) or 7 oligonucleotides (1common primer, 1 common probe, and 5 specific primers).

In some embodiments, the multiplex assay detects 2, 3, 4, 5, 6, or 7 ALKfusions and 2, 3, 4, 5, or 6 RET fusions in a single amplification anddetection reaction. In some embodiments, the multiplex assay furtherdetects 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 ROS1 fusions in thesame reaction. In some embodiments, the ROS1 fusions are detected in aseparate amplification and detection reaction. In some embodiments, theamplification and detection reaction further includes an internalcontrol (e.g., a housekeeping gene).

The presence of ALK, RET and ROS1 fusions indicate that a cancer patientwill be responsive to a selective kinase inhibitor. These includealectinib, crizotinib, ceritinib, lorlatinib, brigatinib, cabozantinib,apatinib, vandetanib, ponatinib, lenvatinib, DS-6051b, and variants orcombinations thereof. The fusion status of a patient can be monitoredthroughout treatment to determine if the therapeutic approach can bechanged, e.g., to a different kinase inhibitor or more standard chemo-or radio-therapy.

IV. Preparation of Sample

Samples for testing for genetic fusions can be obtained from any source,but are advantageously obtained in a non-invasive manner, e.g., fromblood or a blood fraction (e.g., plasma, serum, platelets, etc.).Samples for the present methods can also be taken from urine,bronchoalveolar lavage, or tissue biopsy. Methods for isolating nucleicacids from biological samples are known, e.g., as described in Sambrook,and several kits are commercially available (e.g., High Pure RNAIsolation Kit, High Pure Viral Nudeic Acid Kit, and MagNA Pure LC TotalNucleic Acid Isolation Kit from Roche).

In some embodiments, DNA is prepared, and used as template for thepresently disclosed amplification and detection methods. In someembodiments, RNA is prepared. When RNA is used as template foramplification by PCR, a reverse transcription step is required toprepare cDNA. A DNA polymerase such as Taq or another thermostablepolymerase can then be used to carry out amplification.

In some embodiments, the sample is RNA is isolated from blood plasma.Depending on the condition of the patient, about 1-10 mL of plasma canbe obtained for testing (usually about 2 mL). Kits for isolatingcirculating free RNA are commercially available, e.g., from NorgenBiotek Corp or Qiagen.

As shown in the Examples, the presently disclosed methods for samplepreparation and amplification/detection with custom target-specificoligos are extraordinarily sensitive, and can be used to detect genefusion mutations from as few as about 50—and in some cases about20—copies in a sample diluted 1:4000 in wild type RNA background. Thisallows for detection of fusion variants in samples where the targetsequence is very rare, e.g., circulating cell-free RNA (cfRNA). Varyingbackgrounds of RNA and DNA in plasma do not detract from the specificityof detection even at low copy numbers.

V. Amplification and Detection

Nudeic acid amplification can be carried out using any primer-dependentmethod. In some embodiments, the amplification is quantitative, so thatthe relative or actual abundance of a given amplification target can bedetermined by the amount of amplification product.

DNA-based methods can be used for the presently disclosed amplificationand detection methods, e.g., PCR. In some embodiments, real time orquantitative PCR is used (RTPCR or qPCR). qPCR allows for reliabledetection and measurement of products generated during each cycle of PCRprocess. Such techniques are well known in the art, and kits andreagents are commercially available, e.g., from Roche Molecular Systems,Life Technologies, Bio-Rad, etc. See, e.g., Pfaffl (2010) Methods: Theongoing evolution of qPCR vol. 50. In some embodiments, theamplification and detection are carried out in the presence of a duallabeled probe (e.g., a TaqMan, CPT, LNA, or MGB probe) labeled with aquencher and a fluorophore (see, e.g., Gasparic et al. (2010) Anal.Bioanal. Chem. 396:2023).

In some embodiments, a preliminary reverse transcription step is carriedout (also referred to as RT-PCR, not to be confused with real time PCR).See, e.g., Hierro et al. (2006) 72:7148. The term “qRT-PCR” as usedherein refers to reverse transcription followed by quantitative PCR.Both reactions can be carried out in a single tube without interruption,e.g., to add reagents.

RNA-based amplification methods can also be used, e.g., transcriptionmediated amplification (TMA) or nucleic acid sequence basedamplification (NASBA). See, e.g., Fakruddin et al. (2013) J PharmBioallied Sci. 5:245; van Deursen et al. (1999) Nucl. Acids Res. 27:e15;Kamisango et al. (1999) J Clin. Microbiol. 37:310.

Some of the oligonucleotides used in the present assays (primers andprobes) include alkyl base modifications to enhance selectiveamplification, in particular in a multiplex format.

A probe, or one or both primers in a primer pair can be labeled with anysubstance or component that directly or indirectly emits or generates adetectable signal. In some embodiments, the labels are fluorophores(dyes), many of which are reported in the literature and known to thoseskilled in the art, and many of which are commercially available.Fluorophores are described, e.g., in Cardullo et al. (1988) Proc. Natl.Acad. Sci. USA 85: 8790; Hochstrasser et al. (1992) BiophysicalChemistry 45: 133; Selvin (1995) Methods in Enzymology 246: 300;Steinberg, Ann. Rev. Biochem., 40: 83-114 (1971); and Wang et al., Anal.Chem. 67:

The following are examples of fluorophores that can be used as labels:4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine;acridine isothiocyanate; 5-(2′-aminoethyl)aminonaphthalene-1-sulfonicacid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5disulfonate [0070] N-(4-anilino-1-naphthyl)maleimide; anthranilamide;BODIPY; Brilliant Yellow; coumarin; 7-amino-4-methylcoumarin (AMC,Coumarin 120)/7-amino-4-trifluoromethylcoumarin (Coumaran 151); cyaninedyes; cyanosine 4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin; eosinisothiocyanate; erythrosin B; erythrosin isothiocyanate; ethidium;5-carboxyfluorescein (FAM); dichlorotriazin-2-yl)aminofluorescein(DTAF); 2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE);fluorescein; fluorescein isothiocyanate; fluorescamine; IR144; IR1446;Malachite Green isothiocyanate; 4-methylumbelliferone; orthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;phycoerythrin (including but not limited to B and R types);o-phthaldialdehyde; pyrene; pyrene butyrate; succinimidyl 1-pyrenebutyrate; quantum dots; Reactive Red 4 (Cibacron Brilliant Red 3B-A);6-carboxy-X-rhodamine (ROX); 6-carboxyrhodamine (R6G); lissaminerhodamine B sulfonyl chloride rhodamine; rhodamine B; rhodamine 123;rhodamine X isothiocyanate; sulforhodamine B; sulforhodamine 101;sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;and lanthanide chelate derivatives.

Any of the listed fluorophores (dyes) can be used in the presentlydescribed assays to label a nucleic acid as described herein.Fluorophores can be attached by conventional covalent bonding, usingappropriate functional groups on the fluorophore and/or nucleic acid.

As noted above, a dual labeled probe can be used for detection. The duallabeled probe can comprise a fluorophore, such any of the fluorophoreslisted above, and a quencher. Suitable quenchers include but are notlimited to DDQ-I, Dabcyl, Eclipse, Iowa Black FQ, BHQ-1, QSY-7, BHQ-2,DDQ-II, Iowa Black RQ, QSY-21, and BHQ-3. For fluorophores having anemission maximum between 500 and 550 nm (e.g., FAM, TET, and HEX), aquencher with an absorption maxima between 450 and 500 nm can beselected (e.g., dabcyl or BHQ-1). For fluorophores having an emissionmaximum above 550 nm (e.g., rhodamine and Cy dyes), a quencher with anabsorption maxima above 550 nm can be selected (e.g., BHQ-2). See, e.g.,Johansson (2003) Meth. Mol. Biol. 335:17 for considerations in selectingdye-quencher pairs.

Detection devices are known in the art and can be selected asappropriate for the selected labels. Detection devices appropriate forquantitative PCR include the cobas® and Light Cycler® systems (Roche),PRISM 7000 and 7300 real-time PCR systems (Applied Biosystems), etc.

VI. Kits

In some embodiments, reagents and materials for carrying out thepresently disclosed methods are included in a kit. In some embodiments,the kit includes components for obtaining, storing, and/or preparingsample. Such components include, e.g., sterile needles and syringes,EDTA-lined tubes, buffers (e.g., for binding nucleic acid to, andelution from a matrix), RNase inhibitors, and/or DNase, etc.

In some embodiments, the kit includes forward primer(s) and reverseprimer(s) for amplifying ALK fusion variant(s) having sequences selectedfrom the group consisting of SEQ ID NOs:1-50, and SEQ ID NOs:52-61 and181, respectively. In some embodiments, the kit includes probe(s) fordetecting ALK fusion variant(s) having sequences selected from the groupconsisting of SEQ ID NOs:182-186. The forward and reverse primersequences and probe sequences can be used together in any appropriatecombination to detect any 1, 2, 3, 4, 5, 6, or 7 ALK fusion variants inany combination. In some embodiments, the kit includes forward primer(s)and reverse primer(s) for amplifying RET fusion variant(s) havingsequences selected from the group consisting of SEQ ID NOs:83-145 and187, and SEQ ID NOs:161-180, respectively. In some embodiments, the kitincludes probe(s) for detecting RET fusion variant(s) having sequencesselected from the group consisting of:189-194. The forward and reverseprimer sequences and probe sequences can be used together in anycombination to detect any 1, 2, 3, 4, 5, or 6 RET fusion variants in anycombination. In some embodiments, the kit includes forward primer(s) andreverse primer(s) for amplifying ROS1 fusion variant(s) having sequencesselected from the group consisting of SEQ ID NOs:195-212, and SEQ IDNOs:213-226, respectively. In some embodiments, the kit includesprobe(s) for detecting ROS1 fusion variants having sequences selectedfrom the group consisting of:227-230 and 51. The forward and reverseprimer sequences and probe sequences can be used together in anycombination to detect any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13ROS1 fusion variants in any combination.

In some embodiments, the kit includes a forward primer and reverseprimer for amplifying an EML exon 13-ALK exon 20 fusion variant havingsequences selected from the group consisting of SEQ ID NOs:1-10 and SEQID NOs:52-61 and 181, respectively. In some embodiments, the kitincludes a forward primer and reverse primer for amplifying an EML exon20-ALK exon 20 fusion variant having sequences selected from the groupconsisting of SEQ ID NOs:11-20 and SEQ ID NOs:52-61 and 181,respectively. In some embodiments, the kit includes a forward primer andreverse primer for amplifying an EML exon 6-ALK exon 20 fusion varianthaving sequences selected from the group consisting of SEQ ID NOs:21-30and SEQ ID NOs:52-61 and 181, respectively. the kit includes a forwardprimer and reverse primer for amplifying an EML exon 2-ALK exon 20fusion variant having sequences selected from the group consisting ofSEQ ID NOs:31-35 and SEQ ID NOs:52-61 and 181, respectively. In someembodiments, the kit includes a forward primer and reverse primer foramplifying an EML exon 18-ALK exon 20 fusion variant having sequencesselected from the group consisting of SEQ ID NOs:36-40 and SEQ IDNOs:52-61 and 181, respectively. In some embodiments, the kit includes aforward primer and reverse primer for amplifying a KIF exon 24-ALK exon20 fusion variant having sequences selected from the group consisting ofSEQ ID NOs:41-45 and SEQ ID NOs:52-61 and 181, respectively. In someembodiments, the kit includes a forward primer and reverse primer foramplifying a KIF exon 17-ALK exon 20 fusion variant having sequencesselected from the group consisting of SEQ ID NOs:46-50 and SEQ IDNOs:52-61 and 181, respectively. In some embodiments, the kit includes aprobe for detecting an ALK fusion having a sequence selected from groupconsisting of SEQ ID NOs:182-186.

In some embodiments, the kit includes a forward primer and reverseprimer for amplifying a KIF exon 15-RET exon 12 fusion variant havingsequences selected from the group consisting of SEQ ID NOs:83-97 and SEQID NOs:161-180, respectively. In some embodiments, the kit includes aforward primer and reverse primer for amplifying a KIF exon 16-RET exon12 fusion variant having sequences selected from the group consisting ofSEQ ID NOs:98-107 and SEQ ID NOs:161-180, respectively. In someembodiments, the kit includes a forward primer and reverse primer foramplifying a KIF exon 22-RET exon 12 fusion variant having sequencesselected from the group consisting of SEQ ID NOs:108-117 and SEQ IDNOs:161-180, respectively. In some embodiments, the kit includes aforward primer and reverse primer for amplifying a KIF exon 23-RET exon12 fusion variant having sequences selected from the group consisting ofSEQ ID NOs:118-127 and 187, and SEQ ID NOs:161-180, respectively. Insome embodiments, the kit includes a forward primer and reverse primerfor amplifying a CCDC exon 1-RET exon 12 fusion variant having sequencesselected from the group consisting of SEQ ID NOs:128-135 and 118, andSEQ ID NOs:161-180, respectively. In some embodiments, the kit includesa forward primer and reverse primer for amplifying an NCO exon 6-RETexon 12 fusion variant having sequences selected from the groupconsisting of SEQ ID NOs:136-145 and SEQ ID NOs:161-180, respectively.In some embodiments, the kit includes a probe for detecting a RET fusionhaving a sequence selected from group consisting of SEQ ID NOs:189-194.

In some embodiments, the kit includes a forward primer and reverseprimer for amplifying a CD74 exon 6-ROS1 exon 34 fusion variant havingsequences selected from the group consisting of SEQ ID NOs:195-197 andSEQ ID NOs:222-226, respectively. In some embodiments, the kit includesa forward primer and reverse primer for amplifying a CD74 exon 6-ROS1exon 32 fusion variant having sequences selected from the groupconsisting of SEQ ID NOs:195-197 and SEQ ID NOs:213-215, respectively.In some embodiments, the kit includes a forward primer and reverseprimer for amplifying an EZR exon 10-ROS1 exon 34 fusion variant havingsequences selected from the group consisting of SEQ ID NO:208 and SEQ IDNOs:222-226, respectively. In some embodiments, the kit includes aforward primer and reverse primer for amplifying a TPM3 exon 8-ROS1 exon35 fusion variant having sequences selected from the group consisting ofSEQ ID NOs:211-212 and SEQ ID NOs:216-221, respectively. In someembodiments, the kit includes a forward primer and reverse primer foramplifying an SDC4 exon 4-ROS1 exon 34 fusion variant having sequencesselected from the group consisting of SEQ ID NOs:200-202 and SEQ IDNOs:222-226, respectively. In some embodiments, the kit includes aforward primer and reverse primer for amplifying an SDC4 exon 2-ROS1exon 32 fusion variant having sequences selected from the groupconsisting of SEQ ID NOs:198-199 and SEQ ID NOs:213-215, respectively.In some embodiments, the kit includes a forward primer and reverseprimer for amplifying an SDC4 exon 2-ROS1 exon 34 fusion variant havingsequences selected from the group consisting of SEQ ID NOs:198-199 andSEQ ID NOs:222-226, respectively. In some embodiments, the kit includesa forward primer and reverse primer for amplifying an SDC4 exon 4-ROS1exon 32 fusion variant having sequences selected from the groupconsisting of SEQ ID NOs:200-202 and SEQ ID NOs:213-215, respectively.In some embodiments, the kit includes a forward primer and reverseprimer for amplifying an SLC34A2 exon 13-ROS1 exon 34 fusion varianthaving sequences selected from the group consisting of SEQ IDNOs:203-205 and SEQ ID NOs:222-226, respectively. In some embodiments,the kit includes a forward primer and reverse primer for amplifying anSLC34A2 exon 13-ROS1 exon 32 fusion variant having sequences selectedfrom the group consisting of SEQ ID NOs:203-205 and SEQ ID NOs:213-215,respectively. In some embodiments, the kit includes a forward primer andreverse primer for amplifying an SLC34A2 exon 4-ROS1 exon 32 fusionvariant having sequences selected from the group consisting of SEQ IDNOs:206-207 and SEQ ID NOs:213-215, respectively. In some embodiments,the kit includes a forward primer and reverse primer for amplifying anSLC34A2 exon 4-ROS1 exon 34 fusion variant having sequences selectedfrom the group consisting of SEQ ID NOs:206-207 and SEQ ID NOs:222-226,respectively. In some embodiments, the kit includes a forward primer andreverse primer for amplifying an LRIG3 exon 16-ROS1 exon 35 fusionvariant having sequences selected from the group consisting of SEQ IDNOs:209-210 and SEQ ID NOs:216-221, respectively.

In some embodiments, each of the primer sets is packaged in separatetubes, e.g., to be added in ratios to be determined by the user. In someembodiments, one or more or all of the primer sets are packaged in asingle tube with predetermined ratios.

The kit can also include enzymes, such as reverse transcriptase and orDNA polymerase. In some embodiments, the DNA polymerase is athermostable DNA polymerase capable of amplifying in thermocyclingconditions, e.g., Taq or a Taq derivative. In some embodiments, the kitincludes dNTPs. In some embodiments, the kit includes buffers conduciveto polymerization/amplification by the selected polymerases.

In some embodiments, the kit includes controls, e.g., a polynucleotidethat is wild type at the genetic fusion to be detected (i.e., no geneticfusion), or a polynucleotide that includes the genetic fusion to bedetected.

The kit can also include consumables such as sample tubes or vials;reaction containers (e.g., tubes, multiwell plates, microfluidic chipsor chambers, etc), as well as directions for use or reference to awebsite.

VII. EXAMPLES A. Example 1: Multiplex Assays for Detection of ALK, RET,and ROS1 Fusion Panel

In this example, we tested a multiplex, quantitative RT-PCR method todetect ALK, RET, and ROS1 fusions (ALK/RET/ROS1 panel). Four differentsets of primers and probes are used in a single-tube (or vessel, well,chamber, compartment) assay to reduce the amount of sample needed toachieve measurable, reliable results. These four sets correspond to (i)ALK (detected with one or more probes labeled with a first label), (ii)RET (detected with one or more probes labeled with a second label),(iii) ROS1 (detected with one or more probes labeled with a thirdlabel), and (iv) an internal control (detected with a probe labeled witha forth label). The labels can be selected from those disclosed hereinand in some embodiments are distinguishable from one other. In thepresent example, ALK fusions are detected with a FAM-labeled probe, RETfusions are detected with a HEX-labeled probe, ROS1 fusions are detectedwith a JA270-labeled probe, and the internal control is detected with aCy5.5-labeled probe.

The coverage of the highly multiplexed assay is shown in Table 1 withthe fusion variant number indicated in parenthesis.

TABLE 1 Label Gene Fusion Coverage Oligonucleotides FAM ALK EML4 exon13-ALK exon 20 (V1)  7 fusions 15 primers EML4 exon 20-ALK exon 20 (V2)94% ALK fusions EML4 exon 6a/b-ALK exon 20 (V3) EML4 exon 2-ALK exon 20(V5) EML4 exon 18-ALK exon 20 (V8) KIF5B exon 17-ALK exon 20 (V6) KIF5Bexon 24-ALK exon 20 (V7) HEX RET KIF5B exon 15-RET exon 12 (V1)  6fusions  2 probes KIF5B exon 16-RET exon 12 (V2) 97% RET fusions KIF5Bexon 22-RET exon 12 (V3) KIF5B exon 23-RET exon 12 (V4) CCDC6 exon 1-RETexon 12 (V8) NCOA4 exon 6-RET exon 12 (V9) JA270 ROS1 CD74 exon 6-ROS1exon 34 (V2) 12 fusions 11 primers CD74 exon 6-ROS1 exon 32 (V1) 95%ROS1 fusions  3 probes EZR exon 10-ROS1 exon 34 (V10) TPM3 exon 8-ROS1exon 35 (V13) SDC4 exon 4-ROS1 exon 34 (V5) SDC4 exon 2-ROS1 exon 32(V3) SDC4 exon 2-ROS1 exon 34 (V14) SDC4 exon 4-ROS1 exon 32 (V4)SLC34A2 exon 13-ROS1 exon 34 (V7) SLC34A2 exon 13-ROS1 exon 32 (V6)SLC34A2 exon 4-ROS1 exon 32 (V8) SLC34A2 exon 4-ROS1 exon 34 (V9) LRIG3exon 16-ROS1 exon 35 (V11) CY5.5 IC IC N/A  2 primers  1 probe

The multiplex may include various gene fusion detection combinations,and in some embodiments, fewer fusions are assayed and detected. Anexample of an assay format for detection ALK and RET fusions is shown inTable 2. Fusions in ROS1 can be detected separately, or in a parallelassay, for example, as shown in Table 3.

TABLE 2 Label Gene Fusion Coverage Oligonucleotides FAM ALK EML4 exon13-ALK exon 20 (V1) 7 fusions 15 primers EML4 exon 20-ALK exon 20 (V2)94% ALK fusions EML4 exon 6a/b-ALK exon 20 (V3) EML4 exon 2-ALK exon 20(V5) EML4 exon 18-ALK exon 20 (V8) KIF5B exon 17-ALK exon 20 (V6) KIF5Bexon 24-ALK exon 20 (V7) HEX RET KIF5B exon 15-RET exon 12 (V1) 6fusions  2 probes KIF5B exon 16-RET exon 12 (V2) 97% RET fusions KIF5Bexon 22-RET exon 12 (V3) KIF5B exon 23-RET exon 12 (V4) CCDC6 exon 1-RETexon 12 (V8) NCOA4 exon 6-RET exon 12 (V9) CY5.5 IC IC N/A  2 primers  1probe

TABLE 3 Label Gene Fusion Coverage Oligonucleotides FAM ROS1 CD74 exon6-ROS1 exon 32 (V1) 12 fusions 11 primers SDC4 exon 2-ROS1 exon 32 (V3)95% ROS1 fusions  3 probes SDC4 exon 4-ROS1 exon 32 (V4) SLC34A2 exon13-ROS1 exon 32 (V6) SLC34A2 exon 4-ROS1 exon 32 (V8) HEX ROS1 CD74 exon6-ROS1 exon 34 (V2) EZR exon 10-ROS1 exon 34 (V10) SDC4 exon 4-ROS1 exon34 (V5) SLC34A2 exon 13-ROS1 exon 34 (V7) SLC34A2 exon 4-ROS1 exon 34(V9) SDC4 exon 2-ROS1 exon 34 (V14) JA270 ROS1 TPM3 exon 8-ROS1 exon 35(V13) LRIG3 exon 16-ROS1 exon 35 (V11) CY5.5 IC IC N/A  2 primers  1probe

The oligonucleotides shown in Tables 4-6 can be selected for use in theassays. The first set of forward and reverse primers amplifies acrossEML4-ALK and KIF5B-ALK fusions. The primers are designated with the genename (e.g. EML for EML4), exon (e.g., 13 for exon 13), and designation(e.g., F1 for Forward 1). The symbols <t_bb_dA>, <t_bb_dC>, <t_bb_dT>,<t_bb_dG> refer to p-tert butylbenzyl modified A, C, T, and G,respectively. Forward and reverse primers can be used in single pairs orin any combination to amplify different fusion products, as will beappreciated by one of skill in the art. In the present example, thenumber of oligonucleotides in the reaction was minimized, as indicatedin Tables 1-3. The reverse primers in all reactions served as primersfor the reverse transcriptase reactions.

TABLE 4 Oligonucleotides for use in amplificationand detection of ALK fusions Probe dye SEQ (for ID example) NO SequenceForward  primer FAM EML13F1 1 ACACCTGGGAAAGGACCTAAA EML13F2 2CACACCTGGGAAAGGACCTAAA EML13F3 3 CCACACCTGGGAAAGGACCTA EML13F4 4CCACACCTGGGAAAGGACCT EML13F5 5 CCACACCTGGGAAAGGACC EML13F6 6CCACACCTGGGAAAGGAC EML13F7 7 CCCACACCTGGGAAAGGAC EML13F8 8GCCCACACCTGGGAAAGGA EML13F9 9 AGCCCACACCTGGGAAAG EML13F10 10GAGCCCACACCTGGGAAA EML20F1 11 CTCGGGAGACTATGAAATATTGTACT EML20F2 12TCGGGAGACTATGAAATATTGTACT EML20F3 13 CGGGAGACTATGAAATATTGTACT EML20F4 14CTCGGGAGACTATGAAATATTGTAC EML20F5 15 ACTCGGGAGACTATGAAATATTGTA EML20F616 AACTCGGGAGACTATGAAATATTGTA EML20F7 17 TAACTCGGGAGACTATGAAA TATTGTAEML20F8 18 TAACTCGGGAGACTATGAA ATATTGT EML20F9 19 TAACTCGGGAGACTATGAAATATTGTA EML20F10 20 ACTCGGGAGACTATGAAA TATTGTAC EML6F1 21AAGCATAAAGATGTCATCA TCAACCAA EML6F2 22 AGCATAAAGATGTCATCA TCAACCAAEML6F3 23 GCATAAAGATGTCATCATCAACCAA EML6F4 24 CATAAAGATGTCATCATCAACCAAGEML6F5 25 GCATAAAGATGTCATCATCAACCAAG EML6F6 26 GCATAAAGATGTCATCATCAACCAEML6F7 27 GCATAAAGATGTCATCATCAACC EML6F8 28 AGCATAAAGATGTCATCATCAACCEML6F9 29 AAGCATAAAGATGTCATCATCAACC EML6F10 30 AAGCATAAAGATGTCATCATCAACEML2F1 31 CTCAGTGAAAAAATCAGTCTCAAG EML2F2 32 CTCAGTGAAAAAATCAGTCTCAAGTEML2F3 33 TCAGTGAAAAAATCAGTCTCAAGTA EML2F4 34 TCAGTGAAAAAATCAGTCTCAAGTAAEML2F5 35 CAGTGAAAAAATCAGTC TCAAGTAAAG EML18F1 36 CAGCTCTCTGTGATGCGCTAEML18F2 37 CTCTCTGTGATGCGCTACT EML18F3 38 TCTCTGTGATGCGCTACTCAA EML18F439 GCTCTCTGTGATGCGCTAC EML18F5 40 CTGTGATGCGCTACTCAATAG KIF24F1 41AGAAGAGGGCATTCTGCACA KIF24F2 42 GAGGGCATTCTGCACAGA KIF24F3 43GAGGGCATTCTGCACAGAT KIF24F4 44 GAAGAGGGCATTCTGCACAG KIF24F5 45GGGCATTCTGCACAGATTG KIF17F1 46 GAACTAGTCCAGCTTCGAGCA KIF17F2 47TGAAGAACTAGTCCAGCTTCGA KIF17F3 48 CTAGTCCAGCTTCGAGCACAA KIF17F4 49AAGAACTAGTCCAGCTTCGAG KIF17F5 50 GTCCAGCTTCGAGCACAAG Reverse primerALK20R1 52 GCTCTGCAGCTCCATCTG ALK20R2 53 GGCTCTGCAGCTCCATCT ALK20R3 54GGGCTCTGCAGCTCCATC ALK20R4 55 GGGCTCTGCAGCTCCAT ALK20R5 56GGGCTCTGCAGCTCCA ALK20R6 57 TGCAGCTCCATCTGCATGG ALK20R7 58GCAGCTCCATCTGCATGG ALK20R8 59 CAGCTCCATCTGCATGGC ALK20R9 60AGCTCCATCTGCATGGC ALK20R10 61 GCTCCATCTGCATGGCT ALK20R11 181TGCAGCTCCATCTGCATGGCT TGCAGCTCCATCTGCATGG <t_bb_dC>T Probe ALK20RP9_ 182<DYE-Thr>CCGCCG<BHQ_2> Q6 GAAGCACCAGGAGC ALK20P4 183 <DYE-Thr>TACCGCC<BHQ 2> GGAAGCACCAGGAGCTGCA ALK20P5 184 <DYE-Thr>TACCGCC <BHQ 2>GGAAGCACCAGGAGCTGC ALK20P6 185 <DYE-Thr>TACCGCC <BHQ 2>GGAAGCACCAGGAGCTGALK20P7 186 <DYE-Thr>TACCGCC <BHQ 2>GGAAGCACCAGGAGCT

TABLE 5 Oligonucleotides for use in amplificationand detection of RET fusions Probe dye SEQ (for ID example) NO SequenceForward primer HEX KIF15F1 83 GAATTGCTGTGGGAAATAATGATG KIF15F2 84GAATTGCTGTGGGAAATAATGAT KIF15F3 85 ATTGCTGTGGGAAATAATGAT GTAAAG KIF15F486 TTGCTGTGGGAAATAATGA TGTAAAG KIF15F5 87 TGCTGTGGGAAATAATGATGTAAAGKIF15F6 88 GCTGTGGGAAATAATGATGTAAAG KIF15F7 89 GAATTGCTGTGGGAAATAATGATGTAAA KIF15F8 90 GAATTGCTGTGGGAAATAA TGATGTAA KIF15F9 91AATTGCTGTGGGAAATAATGA TGTAAA KIF15F10 92 ATTGCTGTGGGAAATAATG ATGTAAAKIF15F11 93 ATTGCTGTGGGAAATAATGATGTAA KIF15F12 94AATTGCTGTGGGAAATAATGATGTA KIF15F13 95 ATTGCTGTGGGAAATAATGATGTA KIF15F1496 GAATTGCTGTGGGAAATAA TGATGTA KIF15F15 97 GAATTGCTGTGGGAAATAATGATGTKIF16F1 98 CATGTCAGCTTCGTATCTCTCAA KIF16F2 99 ATGTCAGCTTCGTATCTCTCAAKIF16F3 100 CATGTCAGCTTCGTATCTCTCA KIF16F4 101 GCATGTCAGCTTCGTATCTCTCKIF16F5 102 CATGTCAGCTTCGTATCTCTC KIF16F6 103 GCATGTCAGCTTCGTATCTCTKIF16F7 104 GCATGTCAGCTTCGTATCTC KIF16F8 105 CAGCATGTCAGCTTCGTATCKIF16F9 106 TAGCAGCATGTCAGCTTCGTA KIF16F10 107 AGCAGCATGTCAGCTTCGKIF22F1 108 AGGACCTGGCTACAAGAGTTAA KIF22F2 109 GGACCTGGCTACAAGAGTTAAKIF22F3 110 GGACCTGGCTACAAGAGTTAAA KIF22F4 ill AGGACCTGGCTACAAGAGTTAAAKIF22F5 112 AGGACCTGGCTACAAGAGTTA KIF22F6 113 GGACCTGGCTACAAGAGTTAKIF22F7 114 GACCTGGCTACAAGAGTTAAAAAG KIF22F8 115 ACCTGGCTACAAGAGTTAAAAAGKIF22F9 116 AGGACCTGGCTACAAGAGTT KIF22F10 117 GGACCTGGCTACAAGAGTTKIF23F1 118 TTGAACAGCTCACTAAAGT GCACAAA KIF23F2 119TGAACAGCTCACTAAAGTGCACAAA KIF23F3 120 GAACAGCTCACTAAAGTGCACAAA KIF23F4121 AACAGCTCACTAAAGTGCACAAA KIF23F5 122 ACAGCTCACTAAAGTGCACAAA KIF23F6123 GAACAGCTCACTAAAGTGCACAA KIF23F7 124 AACAGCTCACTAAAGTGCACAA KIF23F8125 ACAGCTCACTAAAGTGCACAA KIF23F9 126 GAACAGCTCACTAAAGTGCACA KIF23F10127 AACAGCTCACTAAAGTGCACA KIF23F13 187 TTGAACAGCTCACTAAAGTGCA CCDC1F1128 TGCGCAAAGCCAGCGT CCDC1F2 129 CGACCTGCGCAAAGCCA CCDC1F3 130GACCTGCGCAAAGCCAG CCDC1F4 131 CCTGCGCAAAGCCAGC CCDC1F5 132ACCTGCGCAAAGCCAGC CCDC1F6 133 CTGCGCAAAGCCAGCGT CCDC1F7 134GACCTGCGCAAAGCCAGC CCDC1F8 135 CGACCTGCGCAAAGCC CCDC1F14 188CAAAGCCAGCGTGACCA NCO6F1 136 TGTATCTCCATGCCAGAGCAG NCO6F2 137GTATCTCCATGCCAGAGCAG NCO6F3 138 CTGTATCTCCATGCCAGAGCA NCO6F4 139GCTGTATCTCCATGCCAGAG NCO6F5 140 GGCTGTATCTCCATGCCAGA GGCTGTATCTCCATGCCAG<t_bb_dA> NCO6F6 141 GGCTGTATCTCCATGCCAG NCO6F7 142 AGGCTGTATCTCCATGCCANCO6F8 143 GAGGCTGTATCTCCATGCCA NCO6F9 144 AGAGGCTGTATCTCCATGC NCO6F10145 GAGAGGCTGTATCTCCATGC Reverse primer RET12R1 161 AGAGTTTTTCCAAGAACCAAGTTCT RET12R2 162 CTAGAGTTTTTCCAAGAAC CAAGTTCT RET12R3 163CTAGAGTTTTTCCAAGAAC CAAGTTC RET12R4 164 CTAGAGTTTTTCCAAGAAC CAAGTTRET12R5 165 CTAGAGTTTTTCCAAGAACCAAGT RET12R6 166 CTAGAGTTTTTCCAAGAACCAAGRET12R7 167 TAGAGTTTTTCCAAGAACCAA GTTCTT RET12R8 168 GAGTTTTTCCAAGAACCAAGTTCTT RET12R9 169 AGTTTTTCCAAGAACCAAGTTCTT RET12R10 170GTTTTTCCAAGAACCAAGTTCTT RET12R11 171 TAGAGTTTTTCCAAGAACCA AGTTCTRET12R12 172 TAGAGTTTTTCCAAGAACC AAGTTC RET12R13 173AGAGTTTTTCCAAGAACCAAGTTC RET12R14 174 AGAGTTTTTCCAAGAACCAAGTT RET12R15175 AGAGTTTTTCCAAGAACCAAGT RET12R16 176 CTCCTAGAGTTTTTCCAAGA ACCAARET12R17 177 CTCCTAGAGTTTTTCCAAGAACCA RET12R18 178TCCTAGAGTTTTTCCAAGAACCAA RET12R19 179 CCTAGAGTTTTTCCAAGAACCAA RET12R20180 GAGTTTTTCCAAGAACCAAGTTCT Probe RET12P3_ 189 <DYE_Thr>ATCCAAA<BHQ_2>HEX GTGGGAATT CCCTCGGAAGAAC RET12P4_ 190 < DYE_Thr>CCAAAGT<BHQ_2> HEXGGGAATT CCCTCGGAAGAAC RET12P8_ 191 < DYE_Thr>TCCAAAG<BHQ_2> HEX TGGGAATTCCCTCGGAAGAA RET12P14_ 192 < DYE_Thr>CCAAAGT<BHQ_2> HEX GGGAATTCCCTCGGAAGAACTT RET12P18_ 193 < DYE_Thr>TCCAAAG<BHQ_2> HEX TGGGAATTCCCTCGGAAGAACTT RET12P13_ 194 < DYE_Thr>ATCCAAA<BHQ_2> HEX GTGGGAATTCCCTCGGAAGAACTT

TABLE 6 Oligonucleotides for use in amplificationand detection of ROSI fusions Probe dye SEQ (for ID example) NO SequenceForward primer JA270 CD74ex6F2 195 CACTGACGCTCCACCGAA CD74ex6F1 196AAGCCCACTGACGCTCCA CD74ex6F3 197 ACTGACGCTCCACCGAAA SDC4ex2F1 198GAGCTGTCTGGCTCTGG <t_BB_dA> SDC4ex2F2 199 TGTCTGGCTCTGGAGATCTTGTCTGGCTCTGGAGAT <t_bb_dC>T SDC4ex4F1 200 TTGAGAGAACGGAGGTCCT SDC4exF2201 TGAGAGAACGGAGGTCCT SDC4ex4F3 202 TTGAGAGAACGGAGGTCCTG SLC34A2ex13F1203 ATAACCATTAGCAGAGAGGCT SLC34A2ex13F2 204 AACCATTAGCAGAGAGGCTCASLC34A2ex13F3 205 ATAACCATTAGCAGAGAGGCT SLC34A2ex4F1 206AGTAGCGCCTTCCAGCT SLC34A2ex4F2 207 GCCTTCCAGCTGGTTGGA EZRex10F2 208GAAGACAAAGAAGGCAGAGAGA LR1G3ex16F1 209 TTCTTACCACAACATGACAGTAGTLR1G3ex16F2 210 TCTTACCACAACATGACAGTAGTG TPM3ex8F1 211GAAAAGACAATTGATGACCTGGA GAAAAGACAATTGATGACCTGG <t_BB_dA> TPM3ex8F5 212AAGCTGGAAAAGACAATTGATGAC Reverse primer ROS1ex32R1 213GTATTGAATTTTTACTCCCTTC TAGTAATTTG ROS1ex32R2 214 GTATTGAATTTTTACTCCCTTCTAGTAATTT ROS1ex32R3 215 GTATTGAATTTTTACTCCCTT CTAGTAATT ROS1ex35R1 216TATAAGCACTGTCACCCCTT ROS1ex35R2 217 ATAAGCACTGTCACCCCTT ROS1ex35R3 218TATAAGCACTGTCACCCCT ROS1ex35R4 219 CTTTGTCTTCGTTTATAAGCA CTGTCAROS1ex35R5 220 AACTCTTTGTCTTCGTTTATAA GCACTGT ROS1ex35R6 221AGCCAACTCTTTGTCTTCGTT TATAAGCA ROS1ex34LArev1 222 CAGTGGGATTGTAACAACCAGAAAT ROS1ex34LArev2 223 GTCAGTGGGATTGTAACAACCAGA ROS1ex34LArev3 224GTCAGTGGGATTGTAACAACCA ROS1ex34LArev4 225 CAGTGGGATTGTAACAACCAGAAAROS1ex34LArev5 226 CAGTGGGATTGTAACAACCAGAA Probe ROS1EX32P2 227<DYE_Thr>TGGAGTCC CAAA<BHQ_2> TAAACCAGGCATTCCCA ROS1EX34P1 228<DYE_Thr>TGATTTT TGGAT<BHQ_2>ACC AGAAACAAGTTTCATAC ROS1EX32P3 229<DYE_Thr>TGGAGTC <BHQ_2>CCAAATAAA CCAGGC<t_BB_dA> TTCCCA ROS1EX34P3 230<DYE_Thr>TGATTT T<BHQ_2>TGGATAC CAGAAACAAGTTTCA TAC ROS1EX35P1 51<DYE_Thr>TCTGGC ATAGAA<BHQ_2>GA TTAAAGAATCAAAAA AGTGCCAAG

We have tested this method using RNA from EML4-ALK positive cell linesNCI-H2228 and EML4-ALK Fusion Variant 1 cell line from HorizonDiscovery, CCDC6-RET cell line LC2AD, as well as from NSCLC formalinfixed paraffin embedded tissue (FFPET) and plasma specimens.

In the case of plasma, we extracted cfRNA using the Roche High PureFFPET RNA extraction kit with MagNA Pure Lysis Buffer and Esperaseenzyme. Because the yield of cfRNA is too low to be measured accurately,we input a fixed volume ( 1/24 of total) of the extracted plasma cfRNAinto the qRT-PCR.

The reaction conditions were as follows. For each reaction, 25 uL ofinput RNA was added to a RT-PCR reaction mix comprising forward andreverse primers, labeled probe, buffer, dUTP, dTTP, dATP, dGTP, UNG, andZ05 enzyme to a final volume of 50 uL. The reactions were run inmultiplex, each with primers and probes specific for every fusionvariant indicated in Table 1.

Results were confirmed using a Next Generation Sequencing assay thatdetects the fusion variants covered in the qRT-PCR assay.

Maximum Ct (threshold cycle) was set at 38, meaning that a signal mustbe detectable over background within a Ct of 38. Data is shown in FIGS.1A, 1B, 1C, and 1D. The input RNA for each reaction was known to havethe indicated fusion variant. Each reaction was repeated three times.

FIG. 1A shows that each ALK fusion variant is detectable at 50 copies.FIG. 1B shows that each RET fusion variant is detectable at 50 copies.FIG. 1C shows that each ROS1 fusion variant is detectable at 50 copies.FIG. 1D shows that the reaction efficiency and input was equivalent, asindicated by the Internal Control Ct's.

B. Example 2: Sensitivity of ALK and RET Fusions in Titered Transcripts

We tested the multiplex qRT-PCR for the limit of detection of the ALKand RET fusion variants shown in Example 1, Table 1. We tested themultiplex assay by titering ALK or RET fusion positive transcripts into0.1 ng Universal Human RNA (UHR) at 250, 100, 50, or 25 copies. Theamplification and detection reactions were repeated 3 times.

As shown in FIG. 2 , all of the ALK and RET fusion variants tested wasdetectable down to 25 copies.

C. Example 3: Linearity Studies and Further Limit of Detection (LOD)Studies

Further studies were carried out to determine the linearity of detectionfor ALK, RET, and ROS1 fusions, as shown and described in FIGS. 3-5 .

Sensitivity, or Limit of Detection (LOD) studies are shown and describedin FIGS. 6-8 . The LOD for each assay is shown in Tables 7 and 8. All 7ALK, 6 RET, and 13 ROS1 fusion variants are detectable down to less than10 copies. The predominant fusion variants are marked with an *.

TABLE 7 LOD for 95% Fusion Hit Rate Probability E13:A20* 12/12 alllevels tested <6.25 copies E20:A20* 12/12 all levels tested <6.25 copiesE6:A20* 12/12 all levels tested <6.25 copies E2:A20 11/12 at 6.25 copies  6.45 copies K17:A20 12/12 at 6.25 copies   4.78 copies (11/12 at 12.5copies) K24:A20 12/12 all levels tested <6.25 copies E18:A20 12/12 alllevels tested <6.25 copies K15:R12* 11/12 at 6.25 copies   6.45 copiesK16:R12* 11/12 at 6.25 copies   6.45 copies K22:R12 12/12 all levelstested <6.25 copies K23:R12 11/12 at 6.25 copies   6.45 copies C1:R12*12/12 all levels tested <6.25 copies N6:R12 12/12 all levels tested<6.25 copies

TABLE 8 Fusion LOD for 95% variant Hit Rate Probability C6:R32 12/12 alllevels tested  <6.25 copies C6:R34* 12/12 all levels tested  <6.25copies SD2:R32 11/12 at 6.25 and 12.5 copies   11.07 copies SD4:R3412/12 all levels tested  <6.25 copies SL13:R32 12/12 all levels tested <6.25 copies SL13:R34 12/12 all levels tested  <6.25 copies SL4:R3212/12 all levels tested  <6.25 copies SL4:R34 11/12 at 6.25 copies   6.45 copies E10:R34* 11/12 at 6.25 copies    6.45 copies L16:R3511/12 at 12.5 copies;    4.78 copies 12/12 at 6.25 copies T8:R35 12/12all levels tested  <6.25 copies SD2:R34 12/12 all levels tested  <6.25copies

While the invention has been described in detail with reference tospecific examples, it will be apparent to one skilled in the art thatvarious modifications can be made within the scope of this invention.Thus the scope of the invention should not be limited by the examplesdescribed herein. All patents, publications, websites, Genbank (or otherdatabase) entries disclosed herein are incorporated by reference intheir entireties.

We claim:
 1. A method of treating an individual with cancer comprising:A. contacting a biological sample from the individual with a compositioncomprising: (i) at least one primer set and probe that specificallyamplify and detect at least one ALK fusion gene, wherein the probe thatspecifically detects the at least one ALK fusion gene is labeled with atleast one non-naturally occurring moiety; (ii) at least one primer setand probe that specifically amplify and detect at least one RET fusiongene, wherein the probe that specifically detects the at least one RETfusion gene is labeled with at least one non-naturally occurring moiety;(iii) at least one primer set and probe that specifically amplify anddetect at least one ROS1 fusion gene, wherein the probe thatspecifically detects the at least one ROS1 fusion gene is labeled withat least one non-naturally occurring moiety; and (iv) a primer and probethat specifically amplify and detect an internal control, wherein theprobe that specifically detects the internal control is labeled with atleast one non-naturally occurring moiety; B. carrying out amplificationand detection under conditions that allow formation and detection of anamplification product in the presence of at least one fusion gene in thebiological sample; C. determining that at least one fusion gene ispresent if a fusion gene is detected in step B; and D. treating theindividual with a kinase inhibitor therapy if at least one fusion geneis present.
 2. The method of claim 1, wherein the at least one ALKfusion gene is selected from the group consisting of: EML4 exon 13-ALKexon 20, EML4 exon 20-ALK exon 20, EML4 exon 6a/b-ALK exon 20, EML4 exon2-ALK exon 20, EML4 exon 18-ALK exon 20, KIF5B exon 17-ALK exon 20, andKIF5B exon 24-ALK exon
 20. 3. The method of claim 1, wherein the atleast one RET fusion gene is selected from the group consisting of:KIF5B exon 15-RET exon 12, KIF5B exon 16-RET exon 12, KIF5B exon 22-RETexon 12, KIF5B exon 23-RET exon 12, CCDC6 exon 1-RET exon 12, and NCOA4exon 6-RET exon
 12. 4. The method of claim 1, wherein the at least oneROS1 fusion gene is selected from the group consisting of: CD74 exon6-ROS1 exon 34, CD74 exon 6-ROS1 exon 32, EZR exon 10-ROS1 exon 34, TPM3exon 8-ROS1 exon 35, SDC4 exon 4-ROS1 exon 34, SDC4 exon 2-ROS1 exon 34,SDC4 exon 2-ROS1 exon 32, SDC4 exon 4-ROS1 exon 32, SLC34A2 exon 13-ROS1exon 34, SLC34A2 exon 13-ROS1 exon 32v2, SLC34A2 exon 4-ROS1 exon 32,SLC34A2 exon 4-ROS1 exon 35, and LRIG3 exon 16-ROS1 exon
 35. 5. Themethod of claim 1, wherein the biological sample includes DNA or RNA. 6.The method of claim 1, wherein the biological sample is RNA from plasmaof the individual.
 7. The method of claim 1, wherein the amplificationand detection are carried out using quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR).
 8. The method of any one of claim1, wherein the kinase inhibitor therapy is selected from the groupconsisting of alectinib, crizotinib, ceritinib, lorlatinib, brigatinib,cabozantinib, apatinib, vandetanib, ponatinib, lenvatinib, DS6051b, or avariant thereof.
 9. A method for determining the presence of at leastone fusion gene in a biological sample from an individual with cancercomprising: A. contacting a biological sample from the individual with acomposition comprising: (i) at least one primer set and probe thatspecifically amplify and detect at least one ALK fusion gene, whereinthe probe that specifically detects the at least one ALK fusion gene islabeled with at least one non-naturally occurring moiety; (ii) at leastone primer set and probe that specifically amplify and detect at leastone RET fusion gene, wherein the probe that specifically detects the atleast one RET fusion gene is labeled with at least one non-naturallyoccurring moiety; (iii) at least one primer set and probe thatspecifically amplify and detect at least one ROS1 fusion gene, whereinthe probe that specifically detects the at least one ROS1 fusion gene islabeled with at least one non-naturally occurring moiety; and (iv) aprimer and probe that specifically amplify and detect an internalcontrol, wherein the probe that specifically detects the internalcontrol is labeled with at least one non-naturally occurring moiety; B.carrying out amplification and detection under conditions that allowformation and detection of an amplification product in the presence ofat least one fusion gene in the biological sample; and C. determiningthe presence of at least one fusion gene if a fusion gene is detected instep B.
 10. The method of claim 9, wherein the at least one ALK fusiongene is selected from the group consisting of: EML4 exon 13-ALK exon 20,EML4 exon 20-ALK exon 20, EML4 exon 6a/b-ALK exon 20, EML4 exon 2-ALKexon 20, EML4 exon 18-ALK exon 20, KIF5B exon 17-ALK exon 20, and KIF5Bexon 24-ALK exon
 20. 11. The method of claim 9, wherein the at least oneRET fusion gene is selected from the group consisting of: KIF5B exon15-RET exon 12, KIF5B exon 16-RET exon 12, KIF5B exon 22-RET exon 12,KIF5B exon 23-RET exon 12, CCDC6 exon 1-RET exon 12, and NCOA4 exon6-RET exon
 12. 12. The method of claim 9, wherein the at least one ROS1fusion gene is selected from the group consisting of: CD74 exon 6-ROS1exon 34, CD74 exon 6-ROS1 exon 32, EZR exon 10-ROS1 exon 34, TPM3 exon8-ROS1 exon 35, SDC4 exon 4-ROS1 exon 34, SDC4 exon 2-ROS1 exon 34, SDC4exon 2-ROS1 exon 32, SDC4 exon 4-ROS1 exon 32, SLC34A2 exon 13-ROS1 exon34, SLC34A2 exon 13-ROS1 exon 32v2, SLC34A2 exon 4-ROS1 exon 32, SLC34A2exon 4-ROS1 exon 35, and LRIG3 exon 16-ROS1 exon
 35. 13. The method ofclaim 9, wherein the biological sample includes DNA or RNA.
 14. Themethod of claim 9, wherein the biological sample is RNA from plasma ofthe individual.
 15. The method of claim 9, wherein the amplification anddetection are carried out using quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR).
 16. The method of claim 1, wherein:(i) the at least one primer set that specifically amplifies the at leastone ALK fusion gene comprises a forward primer comprising SEQ ID NO: 1and a reverse primer comprising SEQ ID NO:52, and the probe thatspecifically detects the at least one ALK fusion gene comprises SEQ IDNO: 182; (ii) the at least one primer set that specifically amplifiesthe at least one RET fusion gene comprises a forward primer comprisingSEQ ID NO: 83 and a reverse primer comprising SEQ ID NO: 161, and theprobe that specifically detects the at least one RET fusion genecomprises SEQ ID NO: 189; and (iii) the at least one primer set thatspecifically amplifies the at least one ROS1 fusion gene comprises aforward primer comprising SEQ ID NO: 195 and a reverse primer comprisingSEQ ID NO: 213, and the probe that specifically detects the at least oneROS1 fusion gene comprises SEQ ID NO:
 227. 17. The method of claim 9,wherein: (i) the at least one primer set that specifically amplifies theat least one ALK fusion gene comprises a forward primer comprising SEQID NO: 1 and a reverse primer comprising SEQ ID NO:52, and the probethat specifically detects the at least one ALK fusion gene comprises SEQID NO: 182; (ii) the at least one primer set that specifically amplifiesthe at least one RET fusion gene comprises a forward primer comprisingSEQ ID NO: 83 and a reverse primer comprising SEQ ID NO: 161, and theprobe that specifically detects the at least one RET fusion genecomprises SEQ ID NO: 189; and (iii) the at least one primer set thatspecifically amplifies the at least one ROS1 fusion gene comprises aforward primer comprising SEQ ID NO: 195 and a reverse primer comprisingSEQ ID NO: 213, and the probe that specifically detects the at least oneROS1 fusion gene comprises SEQ ID NO: 227.